The signal peptidase class of endopeptidases is conserved throughout virtually all cell types, from bacteria to human, and is well known for its canonical role in protein translocation. In bacteria, the type I signal peptidases (SPases) function as part of the general secretory pathway to remove the N-terminal leader sequence that target proteins for translocation across the cytoplasmic membrane. SPase cleavage of preproteins occurs between an N-terminal transmembrane helix and its immediately C-terminal extracellular domain, which frees the mature protein from its membrane-bound post-translocation state. While the identification of SPase substrates remains an area of active research, a general role for SPases in any process other than protein translocation appears never to have been seriously considered. However, there is now experimental evidence of several integral membrane proteins in staphylococci being cleaved by SPase. Using an SPase inhibitor from the arylomycin family of natural products, along with highly sensitive mass spectrometric methods, it is possible to construct dose-response relationships of protein fragments and thereby identify those whose presence in the membrane or the extracellular media decreases with increasing inhibitor concentrations, and thus those that are likely produced via the action of SPase. Using this approach, we have demonstrated that in Staphylococcus aureus and Staphylococcus epidermidis, the arylomycins inhibit the proteolytic cleavage of several polytopic membrane proteins, including OatA, LtaS, and BlaR1, implicating SPase as the responsible endopeptidase. Remarkably, these proteins are not expressed with N-terminal leader sequences, and instead, cleavage occurs at an internal position that topologically resembles a canonical cleavage site because it is located between a transmembrane domain and an immediately C-terminal, soluble extracellular domain. Here, biochemical methods will be used to verify that these proteins are true SPase substrates and a combination of genetic and bioanalytical methods will be used to investigate the biological significance of this non-canonical activity. The generality of the phenomenon will also be investigated through a thorough characterization of the arylomycin-dependent localization of proteins in the periplasm, cell membranes, and extracellular matrix of the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa. The completion of the exploratory Aims of the proposed work is possible within the two year time frame of the R21 funding mechanism and success would validate the non-canonical SPase activities, as well as their biological significance, and would for the first time elucidate a new and unprecedented activity of this putatively well understood class of enzymes. Completion of the proposed experiments will also lay the groundwork for an R01 application focused on the in depth characterization of the non-canonical SPase activities in both Gram-positive and Gram-negative bacteria, and on the extension of these studies to human homologs.